METHOD FOR AUTOMATICALLY CORRECTING AND MATCHING DIGITIZED ELECTROPHORESIS AND WESTERN BLOTS TESTS

Abstract: Two markers of known molecular weight are added to an unknown preparation to be electrophoresed and transferred on membrane surface according to the Western Blot technique. After the sample has been stained the results are imaged by the mean of an opto-electronic scanner. Variations and deformations of the electrophoretic migration are determined by comparing the positions of the markers in the tests to that of the original known relative positions. Based on the changes, an inverse transformation is determined and applied to the digitized image. The transformed images then provide comparable migration results without the degradation produced by uncontrolled factors occuring during the electrophoresis process.

Description: METHOD FOR AUTOMATICALLY CORRECTING AND MATCHING DIGITIZED ELECTROPHORESIS AND WESTERN BLOTS TESTS BACKGROUND OF THE INVENTION This invention concerns a process that makes it possible to automate the calculation of deformations and variations that make possible the matching of electrophoretic separa¬ tion results. The matching and comparison of electrophoretic separation results is traditionally done by the expert's judgement. This method does not make it possible to correct the varia¬ tions and deformations that the electrophoresis technique introduces to the migration pattern of one sample. Some deformations are related to the techniques used to make it possible to separate components of unknown samples. These include deformations related to the physical and electrical actions of the electrophoresis media. Also included is a systematic difference in the electrophoresis step. Also included are the translation and rotation which cannot be reproduced exactly and are related to the placement of the membrane of transfer in case of Western Blot technique after the electrophoresis. SUMMARY OF THE INVENTION The process of the invention makes it possible to correct all these deformations by calculating an inverse transfor¬ mation function and establishing a fixed marker derived from added known labels. The process according to the invention takes place after the preparation's staining stage prior to the expert's ob¬ servation. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a block diagram of a process according to the invention, and a general presentation of the proce¬ dure, Fig. 2 is a perspective view of marking label, and a pre¬ sentation of an electrophoresis gel after separa¬ tion of the preparation, Fig. 3 is a comparison between two profiles prepared from two different gels, and profiles of two reactive tests before matching, and Fig. 4 is a comparison between the two normalised profiles of two different tests after matching. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The process according to the invention includes several steps: (See Fig. 1). I. "Marking" The process according to the invention includes a sample preparation step before an electrophoretic separation is done. A known label containing at least two groups of mole¬ cules of different molecular weight is added to the unknown preparation to be studied. The position of the label will be used as marker points to establish a fixed position and for final calculation of the inverse transformation func¬ tion. 2. "Electrophoresis, transfer and staining" The process according to the invention takes the classic step of making electrophoresis preparation and eventually the transfer onto a membrane using adequate devices. The staining is also done according to the classical techniques i.e. immunoenzymatic or chemical. 3. Diσitation The process according to the invention includes a scanning step with the electro-optical scanner as well as the acqui¬ sition and digitation of images by a computer system. At least one image is acquired for an electrophoresis to be studied. It should contain as required for a scientific ex¬ periment to positive and the negative controls of the expe¬ riment. 4. "Calculation of the Inverse Transformation" The process according to the invention includes a step for calculating the inverse transformation functions making it possible to re-establish more closely from a positive con¬ trol considered a reference a set marker that is fixed for all the tests of the same electrophoresis. This method is as follows: 4.1 Automatic calculation of the apparent migration posi¬ tion of the molecules in the electrophoresis gel contai¬ ning: 4.1.1 Automatic detection of markers by image-ana¬ lysis methods on the image of the negative control and calculation of the position of the summit of the markers profiles on the negative control. 4.1.2 Automatic detection of markers by image-ana¬ lysis methods on the image of the positive control by using the calculations done on the negative con¬ trol. 4.1.3 Calculation of the position of the summit of the numerical profile of the reactions stained on the positive control. 4.1.4 Calculation of the ratio between the position of the summits and comparison with a theoretical coordinate system on the positive control. 4.1.5 Calculation of the correction function to be applied on the tests images by the mean of the cal¬ culation of the correction between the positive control profile and the reference profile. 4.2 Calculation of electrophoresis local deformations (See Fig. 2). Knowing the position of the markers 2 on each stripes and their respective distances, the calculation of the inverse deformation to be applied to the images in order to regi¬ ster the markers and consequently the images that are coor¬ dinated with them uses a matrix model already tested in prior phases which up to now remains sufficient, namely: an affine transformation of the first order with two parame¬ ters. " Recording" An image-recording step is obtained by digital processing applying to the electrophoresis images the series of trans¬ formation functions as well as the local deformation calcu¬ lated by the process in the invention described in the pre¬ ceding step 4. The process according to the invention is particularly ad¬ apted to the reconstruction of the information of immunoen- zymatic reaction from immunodiagnostics tests of "Western- Blot type" observed with an opto-electronic scanner in re¬ flectance or transmission mode. The information can then be used with digital image-processing techniques. The process according to the invention makes is possible to automate the correction of deformations linked with elec¬ trophoresis techniques between different tests coming from different electrophoresis perparations observed with the help of an opto-electronic scanner whose control parameters are computer-controlled. Moreover, the process according to the invention makes it possible to automate the search and comparison of areas on interest on different tests coming from different electro¬ phoresis perparation observed with the help of an opto¬ electronic scanner whose control parameters are computer- controlled.

Claims: CLAIMS 1. A process for automating the matching of electrophoresis results to be scanned and digitized, com¬ prising: - providing a labelling means: using the labelling means to label the specimen to be studied with at least two labels of different molecular weight, - providing an electrophoresis means, - using the electrophoresis means to separate the unknown components of a molecular preparation mixed with the labelling means, - creating an image of the electrophoresis molecular separation either directly or after transfer onto a membrane, - digitizing the image, - calculating the digitized profile of the electro¬ phoresis lane to be studied, - calculating the position of the summit of the mar¬ kers, - calculating an inverse transformation from said known relative positions of the summit and said calculated relative positions, - applying respective inverse transformation functi¬ ons to each lane image to produce a corrected image of each section and producing matched images of the successive lane from said corrected images. 2. A process according to claim 1, wherein the labelling means is represented by at least two known groups of molecules having different molecular weights and being revealed in their positions by a known quantity of specific reactive for these molecules used as marker. 3. A process according to claim 1, wherein the electrophoresis preparation requires a transmission scanner to be digitized. 4. A process according to claim 1, wherein the electrophoresis preparation requires a digitizing means different from a scanner. 5. A process according to claim 2, wherein the marking means is present on only some electrophoresis lane and the inverse transformation calculation are applies by extension on neighbouring lane devoid of markers.

Inventors: BRON CHRISTOPHE R, GODINAUD SYLVIE, LAUNAY DIDIER M, TISSOT MARC, BRON, CHRISTOPHE, R., GODINAUD, SYLVIE, LAUNAY, DIDIER, M., TISSOT, MARC
Applicants: BRON CHRISTOPHE R, GODINAUD SYLVIE, INOPSIA BRON & CO, LAUNAY DIDIER M, TISSOT MARC, BRON, CHRISTOPHE, R., GODINAUD, SYLVIE, INOPSIA BRON & CO., LAUNAY, DIDIER, M., TISSOT, MARC
Application year: 1994
Main classification: G01N27
IPC codes: G01N27